Swine rag-1 gene and utilization thereof

ABSTRACT

BAC clones comprising the porcine RAG-1 gene were obtained by screening a porcine genomic DNA library. Three clones were obtained by subcloning the region comprising the RAG-1 gene from the BAC clones. Genetic maps were obtained by determining the nucleotide sequence of these clones using sequencing, and by mapping using restriction enzyme digests. Based on this information, targeting vectors that can knockout the RAG-1 gene were constructed. Using these vectors it is possible to construct swine that do not possess acquired immunity by suppressing endogenous porcine RAG-1 gene function.

TECHNICAL FIELD

The present invention relates to porcine RAG-1 genes and uses-thereof.

BACKGROUND ART

In vertebrates, the acquired immune system is the major force againstforeign pathogens. The acquired immune system is an antigen-specificimmune condition acquired by the body's immune system as a result ofstimulation by foreign substances (antigens) after birth. The acquiredimmune system is triggered by antigen recognition by T cell antigenreceptors, or immunoglobulins produced by B cells. In the process of Tcell and B cell differentiation, the genes in an embryonic cell genomeare reshuffled and become activated genes, and T cell antigen receptorsand immunoglobulins exert acquired immune activity. Dr. Susumu Tonegawareceived the 1987 Nobel Prize in Physiology and Medicine for thediscovery of gene recombination in the development of the acquiredimmune system.

Gene recombination is initiated by the cleavage of the RecombinationSignal Sequence in genomic genes by the RAG-1 and RAG-2 protein complex(RAG-1 protein is the type 1 product of the Recombination ActivationGene). It is known that in the absence of the RAG-1 gene, T cell antigenreceptors and immunoglobulins cannot be produced, resulting in severecombined immunodeficiency diseases, without acquired immunity. In fact,it is known that RAG-1 gene-deficient mice, obtained by knockoutmethods, cannot produce T cell antigen receptors and immunoglobulins(Mombaerts, P. et al., Cell, (1992) 68 (5), 869-77).

Such mice, which do not possess acquired immunity, can conceivably beused for xenotransplants, retrogenerative medicine, and such. However,there are problems in the application of these mice to such fields,since mouse organs are small compared with human organs, and mice arephysiologically very different from humans (for example, the lifeexpectancy of a mouse is about two years, and mice cells age morerapidly than human cells).

DISCLOSURE OF THE INVENTION

The present inventors considered that swine, whose body fluidcomposition and physiological functions are similar to those of humans,and whose generation time is shorter than that of primates, weresuitably sized animal for application in xenotransplants,retrogenerative medicine, and such. At this time there had been noreports of RAG-1 gene knockout swine. Identifying the porcine RAG-1 genewas prerequisite to constructing a knockout swine, however, there werealso no reports of the isolation of a DNA coding for the porcine RAG-1protein.

The present invention is based on the considerations described above.The first objective of the present invention is to provide DNA codingfor the porcine RAG-1 protein. The present invention also aims toprovide a vector expressing this DNA, transformants in which the vectorhas been introduced, porcine RAG-1 protein and methods for itsproduction, and antibodies which bind to this protein.

Furthermore, an objective of the present invention is to construct swinein which the function of the porcine RAG-1 gene is suppressed. Anotherobjective of the present invention is to provide vectors and cells foruse in constructing these swine.

The present inventors obtained BAC clones comprising the porcine RAG-1gene by screening a porcine genomic DNA library. Regions comprising theRAG-1 gene were subcloned from the BAC clones, and three clones wereobtained. Genetic maps of these clones were constructed by determiningtheir nucleotide sequences using sequencing, and by mapping usingrestriction enzyme digestion. Based on this information, targetingvectors for use in knocking-out the RAG-1 gene were constructed.

Specifically, the region from the KpnI recognition site just before the5′ initiation codon of the RAG-1 gene, to the HincII recognition siteapproximately 1.4 Kb downstream of this KpnI site, was replaced with apuromycin resistance gene. The puromycin resistance gene is used forpositive selection during gene transfer. This substitution prevents theRAG-1 gene from being expressed. The region for homologous recombinationwas the region from the XhoI recognition site approximately 9.0 Kbupstream of the initiation codon, to the XhoI site approximately 2.5 Kbdownstream of the initiation codon. The DT-A gene was also inserted fornegative selection during gene transfer. Swine without an acquiredimmune system can be constructed by suppressing the function of theendogenous RAG-1 gene using the constructed targeting vectors.

Therefore, the present invention relates to DNA coding for swine-derivedRAG-1 protein and uses thereof, especially use for the construction ofswine without an acquired immune system. More specifically, it provides:

-   (1) a porcine-derived DNA of any one of the following (a) to (c):    -   (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or        2;    -   (b) a DNA encoding a protein comprising the amino acid sequence        of SEQ ID NO: 3;    -   (c) a DNA encoding a protein comprising an amino acid sequence        in which one or more amino acids in the amino acid sequence of        SEQ ID NO: 3 have been added, deleted, substituted, and/or        inserted, and also comprising a DNA recombination inducing        activity;-   (2) a vector comprising the DNA of (1);-   (3) a transformed cell containing the DNA of (1), or the vector of    (2);-   (4) a protein encoded by the DNA of (1);-   (5) a method for producing the protein of (4), comprising a step of    culturing the transformed cell of (3), and recovering an expressed    protein from the media of the culture;-   (6) an antibody which binds to the protein of (4);-   (7) a vector comprising a DNA sequence which suppresses the function    of endogenous porcine RAG-1 gene by homologous recombination;-   (8) a porcine cell into which the vector of (7) is introduced;-   (9) a method for producing porcine in which endogenous RAG-1 gene    function is suppressed, comprising the following steps (a) and (b):    -   (a) implanting a nucleus of the cell of (8) into an enucleated        egg;    -   (b) implanting the enucleated egg obtained in (a) into a swine;-   (10) a method for producing a swine with suppressed endogenous RAG-1    gene function, comprising a step of implanting the cell of (8) into    the swine.

The present invention provides DNAs encoding porcine RAG-1 protein. Inthe present invention, “RAG-1 protein” indicates a type 1 product of theRAG protein complex necessary for initiation of gene recombination. Inthe present invention, RAG-1 protein activity can be measured as theactivity of inducing DNA recombination. The activity of inducing DNArecombination can be measured using well known methods (Agawal. A.,Eastmanq. M. and Schatz D. G. (1998) Nature, 394. 744-751). The RAG-1gene sequence and RAG-1 cDNA sequence, which were revealed by thepresent invention, are indicated as SEQ ID NO: 1 and SEQ ID NO: 2,respectively, and the amino acid sequence of the RAG-1 protein encodedby these DNAs is indicated in SEQ ID NO: 3.

The present invention also includes DNAs coding for variants of theporcine RAG-1 protein. Methods well known to those skilled in the artare used to prepare these proteins, and include known methods ofinducing mutations in proteins. For example, one skilled in the art canprepare proteins functionally equivalent to porcine RAG-1 protein (theprotein comprising the amino acid sequence described by SEQ ID NO: 3) byinducing appropriate mutations in to its amino acid sequence usingsite-directed mutagenesis (Hashimoto-Gotoh, T. et al., Gene (1995) 152,271-275; Zoller, M J, and Smith, M., Methods Enzymol. (1983) 100,468-500; Kramer, W. et al., Nucleic Acids Res. (1984) 12, 9441-9456;Kramer W, and Fritz H J., Methods. Enzymol. (1987) 154, 350-367; Kunkel,T A, Proc. Natl. Acad. Sci. USA. (1985) 82, 488-492; Kunkel, MethodsEnzymol. (1988) 85, 2763-2766). Amino acid mutations can also occur innature. The proteins of the present invention also include proteinscomprising the amino acid sequence of porcine RAG-1 protein in which oneor more amino acids are mutated, provided that the resulting mutatedproteins are functionally equivalent to porcine RAG-1 protein. Thenumber of amino acids to be mutated in such mutants is generally 50amino acids or less, preferably 30 amino acids or less, and morepreferably ten amino acids or less (for example, five amino acids orless).

A mutated amino acid residue is preferably mutated to a different aminoacid that conserves the properties of the amino acid side-chain.Examples of the properties of amino acid side chains include hydrophobicamino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D,N, C, E, Q, G, H, K, S, T) and amino acids comprising an aliphaticside-chain (G, A, V, L, I, P), a hydroxyl group-containing side-chain(S, T, Y), a sulfur atom containing side-chain (C, M), a carboxylicacid- and amide-containing side-chain (D, N, E, Q), a base-containingside-chain (R, K, H), or an aromatic-containing side-chain (H, F, Y, W)(The parenthetic letters indicate the one-letter amino acid codes).

Proteins comprising amino acid sequences that are modified from a givenamino acid sequence, by the deletion, addition, and/or substitution withanother amino acid, of one or more amino acid residues, are alreadyknown to retain their biological activity (Mark, D. F. et al., Proc.Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. & Smith, M.,Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci.USA (1982) 79, 6409-6413).

Fusion proteins are included as proteins to which more than one aminoacid residue has been added to the porcine RAG-1 protein amino acidsequence. Fusion proteins are fusions of porcine RAG-1 protein and otherpeptides or proteins, and are included in the present invention. Fusionproteins can be made by techniques well known to those skilled in theart, such as by linking the DNA that encodes porcine RAG-1 protein(comprising the amino acid sequence described in SEQ ID NO: 3) with aDNA encoding another peptide or protein so that their frames match,inserting this fused DNA into an expression vector, and expressing it ina host. There are no restrictions as to the peptides or proteins fusedto the protein of the present invention.

Examples of known peptides that can be used as the other peptide, whichis fused to porcine RAG-1 protein, include FLAG (Hopp, T. P. et al.,Biotechnology (1988) 6, 1204-1210), 6×His containing six His (histidine)residues, 10×His, HA (Influenza agglutinin), human c-myc fragments,VSP-GP fragments, p18HIV fragments, T7-tag, HSV-tag, E-tag, SV40Tantigen fragments, lck tag, α-tubulin fragments, B-tag, Protein Cfragments, and the like. Examples of proteins that may be fused toporcine RAG-1 protein include GST (glutathione-S-transferase), HA(Influenza agglutinin), immunoglobulin constant regions,β-galactosidase, MBP (maltose-binding protein), and such. Fusionproteins can be prepared by fusing commercially available DNAs, whichencode the above fusion peptides or proteins, with a DNA encoding theprotein of the present invention, and expressing the fused DNA thusprepared.

The present invention also provides vectors comprising theabove-mentioned DNAs, transformants in which the vectors have beenintroduced, proteins coded by the above-mentioned DNAs and productionmethod thereof, and antibodies that bind to these proteins.

Vectors generally used by those skilled in the art can be applied to thepresent invention. For example, if E. coli is used as a host fortransformation, it is preferable to use, for example, vectors comprising“ori”, which is required for replication in E. coli, and genes necessaryfor the selection of transformed E. coli (for example, genes resistantto drugs (such as Ampicillin, Tetracycline, Kanamycin, andChloramphenicol)) These vectors include, for example, M13 type vector,pUC type vector, pBR322, pBluescript, pCR-Script, pGEM-T, pDIRECT, andpT7.

An expression vector is especially useful when a vector is used toproduce RAG-1 protein. For example, if aiming for expression in E. coli,expression vectors include vectors comprising a lacZ promoter (Ward etal., Nature (1989) 341, 544-546; FASEB J (1992) 6, 2422-2427), araBpromoter (Better et al., Science (1988) 240, 1041-1043), T7 promoter orsuch. In addition to the above vectors, examples of these kinds ofvectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen),pEGFP, and pET (in this case, the host is preferably T7 RNApolymerase-expressing BL21). Additionally, the expression vectors mayalso contain signal sequences for polypeptide secretion. The pelB signalsequence or the like can be used as the signal sequence for proteinsecretion when, for example, the protein is to be produced in theperiplasm of E. coli (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379).

Other vectors for producing RAG-1 protein include, for example,expression vectors derived from mammals (for example, pcDNA3(Invitrogen) and pEGF-BOS (Nucleic Acids. Res. (1990) 18 (17), p5322),pEF, pCDM8), expression vectors derived from insect cells (for example,“Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8),expression vectors derived from plants (for example pMH1, pMH2),expression vectors derived from animal viruses (for example, pHSV, pMV,pAdexLcw), expression vectors derived from retroviruses (for example,pZIpneo), expression vector derived from yeast (for example, “PichiaExpression Kit” (Invitrogen), pNV11, SP-Q01), and expression vectorsderived from Bacillus subtilis (for example, pPL608, pKTH50).

When aiming to express a vector in animal cells, such as CHO, COS, orNIH3T3 cells, the vector should comprise a promoter necessary forexpression in such cells, for example, SV40 promoter (Mulligan et al.,Nature (1979) 277, 108), MMLV-LTR promoter, the EF1α promoter (Mizushimaet al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, or the like,and preferably comprise a marker gene for selecting transformed cells(for example, genes that are resistant to drugs (e.g., neomycin, G418and such)). Examples of known vectors with these characteristicsinclude, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

A method when aiming to express a gene stably and to amplify the genecopy number in cells is, for example, the introduction of a vectorcomprising a complementary DHFR gene (for example pCHO I) into CHO cellswith a defective pathway for nucleic acid synthesis, and then amplifyingusing methotrexate (MTX). Furthermore, when aiming for transientexpression of a gene, a method can be used wherein a vector comprising aSV40 replication origin (pcD, etc.) is transfected into COS cellscomprising the SV40 T antigen-expressing gene in their chromosomes.

Moreover, the origin of replication can be derived from a polyomavirus,adenovirus, bovine papilomavirus (BPV), and such. Furthermore,expression vectors can contain aminoglycosid transferase (APH) gene,thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolic acid reductase (dhfr), and suchas selective markers for amplification of gene copy number in a hostcell system.

The host cell into which the vector is transfected is not particularlylimited. For example, E. coli and various types of eukaryote cells andsuch can be used. For example, when using eukaryotic cells, animal,plant, or fungi cells can be used as a host. Exemplary animal cellsinclude, for example, mammalian cells such as CHO, COS, 3T3, myeloma,baby hamster kidney (BHK), HeLa, or Vero cells, amphibian cells such asXenopus oocytes (Valle et al. Nature (1981) 291: 340-358), or insectcells such as Sf9, Sf21, or Tn5 cells. Particularly suitable CHO cellsare DHFR-gene-deficient CHO cells dhfr-CHO (Proc. Natl. Acad. Sci.U.S.A. (1980) 77: 4216-4220) or CHO K-1 (Proc. Natl. Acad. Sci. U.S.A.(1968) 60: 1275). Of the animal cells, CHO cells are particularlypreferable when aiming for abundant expression. Nicotianatabacum-derived cells are examples of the plant cells. Known fungi cellsinclude yeast cells such as Saccharomyces, including Saccharomycescerevisiae, or filamentous fungi such as Aspergillus, includingAspergillus niger. When using prokaryotic cells, examples of thebacterial cells include E. coli, for example, JM109, DH5a, HB101,XLlBlue and BL21. In addition, Bacillus subtilis is also known.

When using animals as hosts, mammals, plants, and insects can be used.As the mammals, goats, swine, sheep, mice, and bovine can be used (VickiGlaser, SPECTRUM Biotechnology Applications (1993)). Furthermore, forexample, tobacco can be used when using plants. Silkworms can be used asthe insects, for example.

In order to construct the transformants of the present invention,vectors are introduced into the above-described hosts. Methods forintroducing vectors into host cells such as E. coli include, forexample, calcium chloride methods and electroporation methods (Chu, G.et al., Nucl. Acid. Res. (1987) 15, 1311-1326). When the host cells arecultured cells, vectors can be introduced by, for example, calciumphosphate methods (Chen, C. and Okayama, H. Mol. Cell. Biol. (1987) 7,2745-2752), DEAE Dextran methods (Lopata, M. A. et al., Nucl. Acid. Res.(1984) 12, 5707-5717, Sussman, D. J. and Milman, G. Mol. Cell. Biol.(1985) 4, 1642-1643), cationic liposome DOTAP methods (BoehringerMannheim), and lipofectin methods (Derijard, B. Cell (1994) 7,1025-1037, Lamb, B. T. et al., Nature Genetics (1993) 5, 22-30,Rabindran, S. K. et al., Science (1993) 259, 230-234).

Furthermore, when introducing DNAs into animals, each DNA can beinserted into an appropriate vector (for example, adenovirus vectors(for example pAdexlcw) and retrovirus vectors (for example pZIPneo),without limitation), and introduced into living bodies using methodssuch as retrovirus methods, liposome methods, cationic liposome methods,and adenovirus. Moreover, vectors can be introduced into insects by, forexample, infecting silkworms with a baculovirus comprising a DNA thatencodes a target protein (Susumu, M. et al., Nature (1985) 315,592-594). Moreover, when the DNA is to be introduced into plants, forexample, DNAs encoding target proteins can be inserted into plantexpression vectors such as pMON 530, and introduced into bacteria suchas Agrobacterium tumefaciens. The vectors can be introduced by infectingthese bacteria into tobacco, for example, Nicotiana tabacum (JulianK.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

The RAG-1 proteins of the present invention can be produced, forexample, by culturing transformants of the present invention. Culturescan be carried out according to known methods. For example, culturemedia that can generally be used when culturing animal cells includeDMEM, MEM, RPMI1640, or IMDM. These media may be used with or withoutserum supplements such as fetal calf serum (FCS). The pH of the culturemedium is preferably between about 6 and 8. Such cells are typicallycultured at about 30° C. to 40° C. for about 15 to 200 hours, andculture media may be replaced, aerated, or stirred as necessary.

The RAG-1 proteins of the present invention may be isolated from insideor outside (such as from the medium) of host cells, and can be purifiedas substantially pure homogeneous proteins. General methods for proteinisolation and purification can be used without limitation. For instance,column chromatography, filtration, ultrafiltration, salt precipitation,solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricpoint electrophoresis, dialysis, and recrystallization may beappropriately selected and combined to isolate and purify the protein.Alternatively, protein purification can also be performed by using thesecolumns in multiple combinations.

Examples of chromatography include, for example, affinity chromatographyin which antibodies against RAG-1 proteins of the present invention areimmobilized, ion-exchange chromatography, hydrophobic chromatography,gel filtration, reverse phase chromatography, adsorption chromatography,and such (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed. Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). These chromatographies may be performedby liquid chromatography, such as HPLC and FPLC. By use of thesepurification methods, RAG-1 protein of the present invention can behighly purified.

A protein of the present invention may be optionally modified orpartially deleted by treatment with an appropriate protein modificationenzyme before or after purification. Useful protein modification enzymesinclude, but are not limited to, trypsin, chymotrypsin,lysylendopeptidase, protein kinase, glucosidase, and so on.

When RAG-1 proteins of the present invention are expressed in host cells(for example, animal cells and E. coli) as fusion proteins withGlutathione S-transferase protein, or as recombinant proteins in whichmultiple histidine residues are attached, the expressed recombinantproteins can be purified using a glutathione column or a nickel column.After purifying the fusion proteins, regions other than the targetprotein can be cleaved with thrombin, factor Xa, or such, and removed asnecessary.

Furthermore, proteins can also be recovered from systems in whichindividuals produce a protein. Systems in which proteins are produced byindividual bodies include, for example, production systems using animalsand plants. Proteins can be recovered after introducing target DNAs intoanimals or plants, and producing the proteins in the animal or plantbodies.

For instance, a desired DNA may be prepared as a fusion gene, by fusingit with a gene that encodes a protein specifically produced in milk,such as goat β casein gene. DNA fragments comprising the fusion gene areinjected into goat embryos, which are then impregnated into femalegoats. Target proteins can be recovered from milk produced by thetransgenic goats (i.e., born from goats that received the modifiedembryos) or from their offspring. To increase the amount ofprotein-comprising milk produced by the transgenic goats, appropriatehormones may also be applied to these goats (Ebert K. M. et al.Bio/Technology (1994) 12: 699-702).

When using insects, target proteins can also be recovered from, forexample, the body fluids of silkworms infected with a Baculoviruscomprising a DNA encoding a target protein, (Susumu, M et al., Nature(1985) 315, 592-594).

Furthermore, when using plants, desired proteins can be recovered from,for example, tobacco (tobacco leaves) regenerated using methods wellknown to those skilled in the art (Toki et al., Plant Physiol (1995)100, 1503-1507) from Nicotiana tabacum-derived cells in which a vectorhas been introduced by an above-mentioned method (Julian K.-C. Ma etal., Eur. J. Immunol. (1994) 24, 131-138).

Antibodies which bind to the RAG-1 proteins of the present invention canbe used for purification and detection of RAG-1 protein. Antibodies usedfor the purification and detection of RAG-1 protein in the presentinvention can be produced by known methods. There is no particular limitto the form of the antibodies, which include antiserum obtained byimmunizing immune animals such as rabbits with antigenic protein, allclasses of polyclonal and monoclonal antibodies, as well as humanantibodies and humanized antibodies produced by genetic recombination.Furthermore, antibodies can be antibody fragments and modifiedantibodies as long as they bind to RAG-1 protein.

Antibodies can be obtained by using RAG-1 protein as a sensitizingantigen. RAG-1 proteins for use as sensitizing antigens can be eitherfull-sized proteins or partial peptides of proteins. Moreover,protein-expressing cells or their lysates, or chemically synthesizedRAG-1 proteins can be used as sensitizing antigens. Short peptides canbe used as antigens by properly binding to carrier proteins such askeyhole limpet hemocyanin, bovine serum albumin, or Ovalbumin.

The mammals immunized by sensitizing antigens are not limited, however,they are preferably selected after consideration of their suitabilitywith the parent cells used for cell fusion in monoclonal antibodyproduction. Animals generally used include rodents such as mice,lagomorphs such as rabbits, or primates such as rhesus monkeys. Methodsfor immunizing animals with sensitizing antigens are well known to thoseskilled in the art.

Polyclonal antibodies, for example, may be obtained by collecting bloodfrom antigen-sensitized mammals in which an increase in desiredantibodies in the serum has been confirmed, and separating serum fromthis blood using conventional methods. Serum comprising polyclonalantibodies can be used as the polyclonal antibodies, and fractionscomprising polyclonal antibodies may be further isolated from this serumas necessary.

To prepare monoclonal antibodies, for example, an increase in desiredantibodies is confirmed in the serum of the above-mentionedantigen-sensitized mammals, and then immune cells are collected andsubjected to cell fusion. In this case, spleen cells are specificexamples of immune cells preferably used for cell fusion. Other parentcells to be fused with the above immune cells preferably include, forexample, mammalian myeloma cells, and more preferably myeloma cells thathave acquired a property for selecting fused cells using drugs. Theabove immune cells and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre, G. andMilstein, C., Methods Enzymol. (1981) 73, 3-46).

Next, using known methods, hybridomas that produce the desiredantibodies are cloned from the hybridomas obtained by cell fusion. Thecloned hybridomas are implanted into mouse peritoneal cavities, and thenascitic fluid is recovered from the mice.

Obtained antibodies may be purified to homogeneity. Antibody separationand purification can be performed according to separation andpurification methods used for general proteins. For example, theantibodies may be separated and isolated by the appropriate selectionand combined use of column chromatography such as affinitychromatography, filtration, ultrafiltration, salting-out, dialysis, SDSpolyacrylamide gel electrophoresis, isoelectric focusing, and others(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory (1988)), but these methods are not limiting. Protein Aand protein G columns can be used as columns for affinitychromatography. Exemplary protein A columns include, for example, HyperD, POROS, and Sepharose F.F. (Pharmacia).

For example, the antigen-binding activities of the antibodies can bemeasured by methods such as absorbance measurement, enzyme-linkedimmunosorbent assays (ELISA), enzyme immunoassays (EIA),radioimmunoassays (RIA), and/or immunofluorescence. In ELISA, theantibodies are immobilized on a plate, RAG-1 protein is added to theplate, and then a sample comprising a desired antibody, for example theculture supernatant of antibody-producing cells or purified antibodies,is added. A secondary antibody, which recognizes the primary antibodyand is labeled with an enzyme such as alkaline phosphatase, is thenadded, and the plate is incubated. Next, after washing, an enzymesubstrate such as p-nitrophenyl phosphate is added to the plate, andabsorbance is measured to evaluate the sample's antigen-bindingactivity. Fragments of RAG-1 protein can also be used as a protein thatis added to the plates.

The present invention also provides vectors (targeting vectors) thatcomprise DNA sequences which suppress endogenous porcine RAG-1 genefunction by homologous recombination. Individual swine in whichendogenous RAG-1 gene function is suppressed (the RAG-1 gene is knockedout) can be constructed using these vectors. Suppression of RAG-1 genefunction by such vectors can be performed by suppressing the expressionof the RAG-1 gene, by inducing a mutation in the expression controlregion of the RAG-1 gene. Furthermore, this suppression can be performedby inducing a mutation in the exon region of the RAG-1 gene to express amutant RAG-1 protein, whose activity is suppressed compared with theactivity of wild type RAG-1 protein. “Suppression” of RAG-1 genefunction includes both complete and partial suppression of the RAG-1gene.

The vectors that comprise a DNA sequence which suppresses porcineendogenous RAG-1 gene function by homologous recombination are notlimited, as long as they are able to induce mutations which causesuppression of endogenous porcine RAG-1 gene function. These vectorsinclude, for example, vectors which comprise a mutated expressioncontrol region or exon region of the porcine RAG-1 gene, and/or ahomologous recombination region. Moreover, vectors additionallycomprising a gene for positive selection (selection to choose only cellsin which DNA is introduced) or for negative selection (selection toexclude non-homologous recombinants) are also included. Furthermore,vectors which also comprise a sequence coding for an enzyme whichenhances recombination in the RAG-1 gene site, for example, Cre forCre-lox, are also included.

Induction of mutations can be performed, for example, by using a genefor positive selection as described in the Examples, however, there isno limit to the induction of mutations as long as endogenous RAG-1 genefunction can be suppressed. There is no specific limitation as to thegenes used for positive selection, and examples include puromycinresistance gene and neomycin resistance gene. Genes used for negativeselection include thymidine kinase gene and diphtheria toxin gene.

In the present invention, a homologous recombination region indicates aDNA sequence which mediates homologous recombination when a mutatedexpression control region or exon region of a porcine RAG-1 gene isintroduced into genomic DNA. Therefore, there is no limit to thehomologous recombination region in the present invention, as long as theDNA sequence can mediate homologous recombination. Those skilled in theart can ordinarily carry out selection of DNA sequences.

Moreover, the present invention provides porcine cells in whichtargeting vectors of the present invention are introduced. The porcinecells include somatic cells, fertilized eggs, and such, but are notlimited thereto. In the present invention, methods known to thoseskilled in the art can be used to introduce targeting vectors into theabove-mentioned cells. Specifically, electroporation methods can beperformed. For example, 1×10⁷ cells and 50 nM targeting vector aresuspended in 500 μl HEPES buffer and transferred into a 4.0 mm gappedelectroporation cuvette. A gene is introduced into the cell by applyingone pulse using an electroporator at a setting of 250V, 960 μF, andinfinite resistance (values can vary depending on cell origin).

Moreover, the present invention provides methods to produce RAG-1 geneknockout swine (endogenous RAG-1 gene function is suppressed). Thesemethods are not particularly limited, and for example include methodswhere an enucleated egg is implanted with a nucleus from a somatic cellin which endogenous RAG-1 gene function is suppressed, and the obtainedenucleated egg is then implanted into swine, which can give birth toindividual swine. Other methods include methods of directly injecting atargeting vector of the present invention into fertilized eggs, and thenimplanting the fertilized eggs into swine, which can give birth toindividual swine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a vector in which the RAG-1 genehas been subcloned. K, X, S, H, B, and N indicate KpnI, XhoI, SacI,HincII, BamHI, and NotI, respectively. (a) indicates genomic DNA, (b)indicates RAG-1-BAC-SacI, (c) indicates Rag1-Clawn-genome-BamHI, (d)indicates pBKS-RagKp.

FIG. 2 is a schematic illustration of a genetic map of the RAG-1 generegion. The diagonal box (1) indicates the first exon, diagonal box (2)indicates the second exon, and the region between (1) and (2) indicatesan intron. The thick line (3) indicates the region used for the newlydevised probe (e.g. RAG-1 Koitabashi probe) for cloning. The thick line(4) indicates the region used as a probe for subcloning from BAC (intron5′ upstream probe). The region between (A) and (B) indicates theClawn-genome-BamHI subcloning site, and the region between (C) and (D)indicates the BAC-SacI subcloning site.

FIG. 3 is a schematic illustration of the targeting vector and a geneticmap of the RAG-1 gene region before and after RAG-1 targeting. K, X, S,H, B, Sal, and N indicate Kpn, XhoI, SacI™ HincII, BamHI, SalI, andNotI, respectively. (a) indicates genomic DNA, (b) indicates a targetingvector, (c) indicates a target genome.

FIG. 4 is a schematic illustration of the process of constructing theRAG-1 gene targeting vectors. K, X, S, H, B, Sal, and N indicate KpnI,XhoI, SacI, HincII, BamHI, SalI, and NotI respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is specifically illustrated below with referenceto Examples, but it is not to be construed as being limited thereto.

EXAMPLE 1 Construction of a Targeting Vector for Porcine RAG-1 GeneKnockout

First, the isolation of porcine RAG-1 cDNA was attempted. At this time,known cDNA could not be obtained. Furthermore, after detailedinvestigation of information on known mouse and human nucleotidesequences, it was determined that porcine RAG-1 cDNA might notcross-hybridize with probes derived from other species. Therefore,reverse transcription PCR from porcine mRNA was performed. First, fourPCR primers known for their use for the human RAG-1 gene were used (Chunet al., Cell (1991) 64: 189-200); however, PCR product from swine wasnot obtained. Therefore, as the result of the trial and error productionof a large number of primers, using mice and human nucleotide sequenceconsensus regions as leads, a 1161 bp cDNA fragment (the region from7010 bp to 8171 bp in the attached 9.1 Kb nucleotide sequence) with 88%homology to the human RAG-1 cDNA nucleotide sequence was cloned. Aporcine thymus cDNA library was screened using this cDNA fragment as aprobe, and a cDNA clone covering about 80% of the full-length wasobtained. The rest of the cDNA nucleotide sequence was obtained using 5′and 3′ RACE methods. Thus, cloning porcine RAG-1 cDNA in sections wasextremely difficult.

Next, PCR primers were constructed based on the information obtainedfrom this cDNA. A porcine genomic DNA library was screened using theconstructed PCR primers, and BAC clone 578B12, which comprises theporcine RAG-1 gene, was obtained. The region of the BAC clone thatcomprised the RAG-1 gene was subcloned, and three clones (approximately6.1 Kb Rag1-BAC-SacI, approximately 6.0 Kb Rag1-Clawn-genome-BamHI, andapproximately 12.6 Kb pBKS-RagKp) were obtained (FIG. 1). The nucleotidesequences of these clones were determined by sequencing (approximately9.1 Kb) (SEQ ID NO: 1), and were mapped by restriction enzyme digests inorder to construct an approximately 16 Kb genetic map (FIGS. 1 and 2).The targeting vectors for knockout of the RAG-1 gene were constructedbased on this information.

Using this targeting vector, the region from the KpnI recognition site,located immediately 5′ of the RAG-1 gene initiation codon, to the HincIIrecognition site, located approximately 1.4 Kb downstream from the KpnIsite, was replaced with a puromycin resistance gene (FIG. 3). Thispuromycin resistance gene is utilized for positive selection during genetransfer. The RAG-1 gene cannot be expressed because of thissubstitution. The region for homologous recombination is defined as theregion between the XhoI recognition site, located approximately 9.0 Kbupstream of the initiation codon, and the XhoI recognition site, locatedapproximately 2.5 Kb downstream of this initiation codon. Furthermore,the DT-A gene is linked for use for negative selection during genetransfer (FIG. 3).

First, pBKS-RagKp, which comprised the 5′ side of the homologousrecombination region, was digested with XhoI and self-ligated to obtainpBKS-RagXK. Furthermore, Rag1-Clawn-genome-BamHI, which comprised the 3′side of the homologous recombination region, was digested with HincIIand NotI to obtain an approximately 2.2 Kb fragment. pBKS-RagHN wasobtained by ligating this fragment to pBluescriptKS(−), which had beendigested with HincII and NotI in the same way. This vector was thenpartially digested with XhoI and self-ligated to obtain pBKS-RagHX, inwhich the approximately 1.2 Kb comprising the stop codon was removed.The approximately 4.0 Kb fragment obtained by partially digestingpBKS-RagHX with XhoI, was ligated to the approximately 1.7 Kb fragmentobtained by digesting pPGK-puro, which comprised the puromycinresistance gene, with SalI. Thus, two different types of vectors withopposite orientation of the puromycin resistance gene were obtained:pBKS-PuroF-RagHX and pBKS-PuroR-RagHX. These two vectors were digestedwith KpnI and NotI, and the approximately 2.7 Kb fragment thus obtainedwas linked with the approximately 9.0 Kb fragment obtained by digestingthe above-mentioned pBKS-RagXK with SalI and KpnI, and an approximately4.3 Kb fragment obtained by digesting a pMClDTpAMCS vector comprisingthe DT-A gene with NotI and SalI. The targeting vectors pTV-Rag1puroFand pTV-Rag1puroR were thus obtained (FIG. 4).

Industrial Applicability

Swine without T cell antigen receptors and immunoglobulins can beobtained using the targeting vectors of the present invention. Thisresults in swine that lack acquired immunity, rendering severalimportant applications possible.

(1) Miniature Swine for Xenotransplants

At the present time, due to advances in technology, the demand fororgans for organ transplant from brain-dead individuals is more thanfive times that of supply. Therefore, there is a serious undersupply oforgans, such that there is even an organ black market. Modifiedminiature swine that carry human genes for organ transplant into humanshave already been developed by several European and American venturecompanies, and the transplanted condition has been successfullymaintained for several months in primates such as baboons. In theseminiature swine for use in xenotransplants, rejection that arises at arelatively early stage, such as hyperacute vascular rejection, does notoccur. However, in order to maintain transplants for a long period,cellular rejection, which appears gradually, must be understood. Twoaspects of the problem of cellular rejection must be examined: that ofhow a transplant recipient's acquired immune system reacts with thetransplanted organ; and the problem of runaway porcine immune function,where the porcine T cells and B cells attached to the transplanted organproliferate in the transplant recipient's body, and recognize therecipient as a foreign object. The RAG-1 gene in miniature swine forxenograft use is knocked out, and post-transplant transfer of theporcine acquired immune system to recipients does not occur in swinewithout T cells and B cells. As a result, the success rate ofxenotransplantation is expected to rapidly increase.

(2) RAG-1 Knockout Swine as Human Organ Incubators for RetrogenerativeMedicine

Nude mice lacking a thymus do not possess acquired immunity, due to Tcell antigen receptor deficiency, and hence they do not reject humancells and organs. Therefore, transplant organs for plastic surgery, suchas human noses and auricles, have been cultured in nude mice. However,mice are limited in size, and larger organs for retrogenerative medicineuse cannot be produced.

The body fluid composition and physiological function of omnivorousswine is more similar to humans than that of other herbivorous livestockanimals. Swine also have a shorter generation time than primates. Thus,swine are the most suitable animals to use as incubator bodies forculturing human organs for retrogenerative medicine. Since RAG-1knockout swine have lost their acquired immunity, they are similar tonude mice in that they do not reject human organs, and can supplynutrients and culture organs.

(3) Human Acquired Immunity Developed using Swine

It is predicted that human acquired immunity can be established bytransferring human bone marrow into RAG-1 knockout swine, since they donot have their own acquired immunity. If porcine/human bone marrowchimeric animals for acquired immunity can be developed, it will nolonger be necessary to humanize mouse antibodies or the like usingconventional recombinant DNA techniques. It will be possible to obtainswine that directly produce human antibodies. With regards to T cells,Human and swine MHC (major histocompatibility antigen complex) class IImolecules, which have an important role in T cell antigen recognition,are very similar, and human helper T cells that function in humans cantherefore be produced. For example, AIDS virus infections are mediatedby the CD4 molecule of helper T cells, causing the death of helper Tcells. As a result, AIDS-infected patients lose their acquired immunityand die from opportunistic infections. However, CD4 molecules thatresist the human AIDS virus have been already constructed. Therefore,acquired immunity can be restored to AIDS patients by making theirhelper T cells resistant to the AIDS virus, amplifying these cells inRAG-1 knockout swine, and then transferring the cells back into thepatients. Immunity and immune activity can be recovered in patients withimmunodeficiency, or with reduced immunity due to aging, by injection oftheir own acquired immune system cells, which have been amplified insidethe body of RAG-1 knockout porcine.

1. A porcine-derived DNA of any one of the following (a) to (c): (a) aDNA comprising the nucleotide sequence of SEQ ID NO: 1 or 2; (b) a DNAencoding a protein comprising the amino acid sequence of SEQ ID NO: 3;(c) a DNA encoding a protein comprising an amino acid sequence in whichone or more amino acids in the amino acid sequence of SEQ ID NO: 3 havebeen added, deleted, substituted, and/or inserted, and also comprising aDNA recombination inducing activity.
 2. A vector comprising the DNA ofclaim
 1. 3. A transformed cell containing the DNA of claim 1, or thevector of claim
 2. 4. A protein encoded by the DNA of claim
 1. 5. Amethod for producing the protein of claim 4, comprising a step ofculturing the transformed cell of claim 3, and recovering an expressedprotein from the media of the culture.
 6. An antibody which binds to theprotein of claim
 4. 7. A vector comprising a DNA sequence whichsuppresses the function of endogenous porcine RAG-1 gene by homologousrecombination.
 8. A porcine cell into which the vector of claim 7 isintroduced.
 9. A method for producing porcine in which endogenous RAG-1gene function is suppressed, comprising the following steps (a) and (b):(a) implanting a nucleus of the cell of claim 8 into an enucleated egg;(b) implanting the enucleated egg obtained in (a) into a swine.
 10. Amethod for producing a swine with suppressed endogenous RAG-1 genefunction, comprising a step of implanting the cell of claim 8 into theswine.